The present invention relates generally to immunotherapeutic agents or therapeutic agents, compositions comprising those agents, and methods of use of those agents and compositions for the treatment and clinical remission of psoriasis.
Psoriasis is a chronic, genetically-influenced, remitting and relapsing scaly and inflammatory skin disorder of unknown etiology that affects 1 to 3 percent of the world""s population. There are several types of psoriasis, including plaque, pustular, guttate and arthritic variants. There is at present no cure for psoriasis, but rather only suppressive therapy (Greaves and Weinstein, 1995, Drug Therapy, 332: 581-588). Indications for treatment may arise from local symptoms, for example, pain, itching, reduction of manual dexterity, severe problems with walking, and cosmetic problems such as prominent hand, leg, or facial lesions. Due to the toxicity of the available therapies, patients with limited disease will often decide to forego treatment beyond the avoidance of provoking factors.
The goal of current treatments has been to decrease the severity and extent of psoriasis to the point at which it no longer interferes substantially with the patient""s occupation, well-being, or personal or social life. The initial treatment for stable plaque psoriasis of any severity is topical. In patients in which more than 20 percent of the skin is affected, however, topical treatment alone may be impractical and systemic therapy may also be indicated at the outset.
The topical treatment for plaque psoriasis incorporates the use of emollients, keratolytic agents, coal tar, anthralin, corticosteroids of medium to strong potency, and calpotriene. All of these treatments have variable efficacy, fail to prevent frequent relapses of the disease, exhibit side effects, and pose cosmetic problems of their own.
Systemic treatment has been used in patients with physically, socially, or economically disabling psoriasis that has not responded to topical treatment. The choices to date have been phototherapy or systemic drug therapy. Generally, systemic treatment has employed phototherapy with Ultraviolet B irradiation, photo chemotherapy which combines the photosensitizing drug methoxsalen with Ultraviolet A phototherapy (PUVA), methotrexate, etretinate, systemic corticosteroids, and cyclosporine. Each of these systemic treatments has variable efficacy and undesired side effects, and some of them are very toxic and present frequent relapses of the disease. Accordingly, there is at present a need for an effective psoriasis treatment that avoids the disadvantages associated with the currently available topical or systemic treatments.
The present invention provides compositions and methods for the treatment and clinical remission of psoriasis. In one aspect, polypeptides derived from particulate antigens of protozoans of the genus Leishmania are provided that can generate an immune is response in an individual resulting in abatement of the clinical symptoms of psoriasis. In one embodiment of this aspect, the polypeptides comprise at least one of the amino acid sequences recited in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, or immunogenic variants thereof. An immunogenic variant of an amino acid sequence may be either a truncated version of the sequence that retains a substantial amount of the activity of the original, or an altered version of the sequence retaining such activity and having conservative amino acid substitutions and/or modifications.
In another embodiment of this aspect, the polypeptides comprise at least an immunogenic portion of an amino acid sequence of a protein, or an immunogenic variant thereof, the protein isolated from protozoans of the genus Leishmania and having an apparent molecular weight of 21, 33, 44, 50, 55, 58, 65, or 77 kDa as determined by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
In yet another embodiment of this aspect, the polypeptides comprise at least an immunogenic portion of an amino acid sequence of a protein, or an immunogenic variant thereof, the protein isolated from protozoans of the genus Leishmania and having an apparent molecular weight after total reduction and alkylation of 73, 80, or 82 kDa as determined by SDS-PAGE.
In related aspects, the present invention provides nucleic acid sequences encoding such polypeptides, vectors incorporating such nucleic acid sequences, methods for the production of such polypeptides by transformation, transfection, or transduction of microbial host cells, and microbial host cells transformed by such vectors, transfected by such nucleic acid sequences, or transduced with such nucleic acid sequences.
In another aspect of the invention, immunotherapeutic agents incorporating one or more polypeptides of the present invention are provided. These immunotherapeutic agents may be polyvalent or monovalent, and may incorporate an adjuvant such as alumina to enhance the immune response obtained from an inoculated subject. In a preferred embodiment, the polyvalent immunotherapeutic agent contains polypeptide isolates from a mixture of four species of Leishmania, namely, L.(L)amazonensis, L.(L)venezuelensis, L.(V)brasiliensis, and L.(L)chagasi. Also in a preferred embodiment, the monovalent immunotherapeutic agent contains a polypeptide isolate from one of these four species.
In another aspect of the invention, methods for the treatment and clinical remission of psoriasis are provided. In one embodiment, such a method involves administration of a therapeutically effective amount of a pharmaceutical composition comprising one or more polypeptides of the present invention to a subject in order to induce an immune response resulting in abatement of the clinical symptoms of psoriasis. In a related embodiment, the method involves administration of a therapeutically effective amount of a pharmaceutical composition comprising one or more nucleic acid sequences of the present invention to a subject. The term xe2x80x9ctherapeutically effectivexe2x80x9d as used herein means a reduction of approximately 70-100% of Psoriasis Area and Severity Index as is understood by those skilled in the art.
Other aspects of the invention include the use of the nucleic acid sequences of the invention as probes for genetic analysis and as nucleic acid molecular weight markers, and the use of the polypeptides of the invention as molecular weight markers.
The present invention concerns novel compositions and methods for the treatment and clinical remission of psoriasis. The compositions comprise immunogenic polypeptides or the nucleic acids encoding them. The polypeptides of the present invention can elicit an immune response in a warm-blooded animal, thereby inducing clinical remission of psoriasis. As used herein, the term xe2x80x9cwarm-blooded animalxe2x80x9d includes humans. In one embodiment of the invention, the subject polypeptides can be isolated from Leishmania protozoa and, preferably, from killed Leishmania amastigote protozoa. The polypeptides of the subject invention can be obtained from protozoa of the Leishmania genus using standard protein isolation procedures which are known in the art. Also contemplated by the present invention are immunotherapeutic agents and pharmaceutical compositions incorporating the immunogenic polypeptides of the present invention. In one embodiment, a first-generation polyvalent immunotherapeutic agent is provided, comprising a polypeptide isolate of a mixture of a plurality of Leishmania species, such as L.(L)amazonensis, L.(L)venezuelensis, L.(V)brasiliensis, L.(L)chagasi, L.(L)donovani, L.(L)infantum, L.(L)major, L.(L)panamensis, L.(L)tropica, and L.(L)guyanensis. Preferably, the mixture comprises L.(L)amazonensis, L.(L)venezuelensis, L.(V)brasiliensis, and L.(L)chagasi. Most preferably, the mixture consists of these four species. The organisms are preferably cultivated in the amastigote stage in the synthetic culture medium specified in Table 1, supplemented with 5% fetal bovine serum, typically at about 30-34xc2x0 C. Subsequently, and during the stationary phase of growth, the amastigotes are subjected to a medium containing an amount of N-p-tosyl-L-Lysine chloromethyl ketone (TLCK) or a pharmacologically acceptable salt thereof effective to kill the cells. The dead cells are then isolated and treated with the non-ionic detergent Nonidet p-40 (NP40) to solubilize the surface antigens, which are discarded. The particulate antigens that comprise the immunogenic polypeptides of the present invention can be collected by centrifugation following cell disruption. These polypeptides are washed with phosphate-buffered saline (PBS) and subsequently resuspended by sonication for 5 minutes at 4xc2x0 C. in PBS containing alumina.
In another embodiment, a first-generation monovalent immunotherapeutic agent is described, comprising a polypeptide isolate of a single Leishmania species chosen from the group consisting of L.(L)amazonensis, L. (L)venezuelensis, L.(V)brasiliensis, L.(L)chagasi, L.(L)donovani, L.(L)infantum, L.(L)major, L. (L)panamensis, L.(L)tropica, and L.(L)guyanensis. Preferably, the single Leishmania species is chosen from the group consisting of L.(L)amazonensis, L.(L)venezuelensis, L.(V)brasiliensis, and L.(L)chagasi. Procedures for the preparation of this immunotherapeutic agent are otherwise identical to those disclosed above for the first-generation polyvalent immunotherapeutic agent.
In another embodiment, a second-generation polyvalent immunotherapeutic agent is described, comprising a polypeptide isolate of a mixture of a plurality of Leishmania species, such as L.(L)amazonensis, L.(L)venezuelensis, L. (V)brasiliensis, L.(L)chagasi, L.(L)donovani, L.(L)infantum, L. (L)major, L.(L)panamensis, L.(L)tropica, and L.(L)guyanensis. Preferably, the mixture comprises L.(L)amazonensis, L.(L)venezuelensis, L.(V)brasiliensis, and L.(L)chagasi. Most preferably, the mixture consists of these four species. The organisms are preferably cultivated in the amastigote stage in the synthetic culture medium specified in Table 1, supplemented with 5% fetal bovine serum, typically at about 30-34xc2x0 C. Subsequently, and during the stationary phase of growth, the amastigotes are subjected to a medium containing an amount of N-p-tosyl-L-Lysine chloromethyl ketone (TLCK) or a pharmacologically acceptable salt thereof effective to kill the cells. The dead cells are then isolated and treated with the non-ionic detergent Nonidet p-40 (NP40) to solubilize the surface antigens, which are discarded. The particulate antigens that comprise the immunogenic polypeptides of the present invention can be collected by centrifugation following cell disruption. These polypeptides are washed with phosphate-buffered saline (PBS) and subsequently resuspended by sonication for 5 minutes at 4xc2x0 C. in 8 M Urea, 0.025 M Tris (Tris-hydroxy-methyl-amino-methane). The polypeptides are then subjected to chromatography on a DEAE-Sephadex column with a stepwise elution from 0.05-0.3 M NaCl in a solution containing 8 M Urea, 0.025 M Tris, pH 8.3. Seven protein fractions are collected, and an inoculum comprising each protein fraction is made by resuspending the polypeptides of each fraction in PBS containing alumina.
In another embodiment, a second-generation monovalent immunotherapeutic agent is described, comprising a polypeptide isolate of a single Leishmania species chosen from the group consisting of L.(L)amazonensis, L.(L)venezuelensis, L.(V)brasiliensis, L.(L)chagasi, L.(L)donovani, L.(L)infantum, L.(L)major, L.(L)panamensis, L.(L)tropica, and L.(L)guyanensis. Preferably, the single Leishmania species is chosen from the group consisting of L.(L)amazonensis, L.(L)venezuelensis, L.(V)brasiliensis, and L.(L)chagasi. Procedures for the preparation of this immunotherapeutic agent are otherwise identical to those disclosed above for the second-generation polyvalent immunotherapeutic agent.
Alternatively, the subject polypeptides can be synthesized according to known procedures and techniques, or produced recombinantly by taansforming a host cell with one or more of the nucleotide sequences encoding the desired polypeptides. The polypeptides can be expressed in the host cell such that they can be isolated and purified to a desired degree of purification. The subject polypeptides can be used in accordance with the subject invention as a third-generation immunotherapeutic agent to treat psoriasis.
The instant invention further concerns nucleic acid sequences that can be useful in transforming appropriate host cells to cause them to produce the polypeptides of the invention; in administration to a warm-blooded animal, either directly or as part of a pharmaceutically-acceptable composition, to generate an immune response and thereby induce clinical remission of psoriasis in the animal; as labelled probes for genetic analysis; or as nucleic acid molecular weight markers.
One of ordinary skill in the art of molecular biology can obtain nucleic acids encoding the polypeptides of the present invention in view of the teachings provided herein. For example, the polypeptides of the first-generation immunotherapeutic agent of the present invention have been isolated and purified from protozoa of the Leishmania genus and comprise eight bands, identified by SDS-PAGE, representing eight distinct polypeptides having apparent molecular weights of 21, 33, 44, 50, 55, 58, 65, and 77 kDa, respectively. Each of these bands represents a separate polypeptide that can be isolated and sequenced in accordance with standard amino acid sequencing procedures. The polypeptides of each second-generation immunotherapeutic agent were purified by subjecting the first-generation immunotherapeutic agent containing the mixture of eight polypeptides to chromatography on diethylaminoethyl(DEAE)-Sephadex. Two fractions having all the activity to cure psoriasis were isolated and totally reduced and alkylated by standard procedures. These fractions were subjected to electrophoresis on acrylamide gels to separate the constituent polypeptides, and the amino acid sequence of each polypeptide was obtained by standard protein sequencing procedures. The nucleotide sequences encoding each of these polypeptides can be derived from these amino acid sequences by application of the genetic code.
Additionally, the present invention contemplates the production of large quantities of the immunogenic polypeptides of the invention via introduction of the nucleic acids encoding them to microbial host cells. The nucleic acids can be introduced directly into the genome of the host cell or can first be incorporated into a vector which is then introduced into the host. Exemplary methods of direct incorporation include transduction by recombinant phage or cosmids, transfection where specially treated host bacterial cells can be caused to take up naked phage chromosomes, and transformation by calcium precipitation. These methods are well known in the art.
Exemplary vectors include plasmids, cosmids, and phages. A genomic library for a Leishmania species can be created by routine means, and DNA of interest isolated therefrom. For example, DNA of Leishmania protozoa can be isolated and restricted with known restriction enzymes. The resulting DNA fragments can then be inserted into suitable cloning vectors for introduction to a compatible host. Depending on the contemplated host, the vector may include various regulatory and other regions, usually including an origin of replication, one or more promoter regions, and markers for the selection of transformants. In general, the vectors will provide regulatory signals for expression and amplification of the DNA of interest.
Various markers may be employed for the selection of transformants, including biocide resistance, particularly to antibiotics such as ampicillin, tetracycline, trimethoprim, chloramphenicol, and penicillin; toxins, such as colicin; and heavy metals, such as mercuric salts. Alternatively, complementation providing an essential nutrient to an auxotrophic host may be employed.
Hosts which may be employed according to techniques well known in the art for the production of the polypeptides of the present invention include unicellular microorganisms, such as prokaryotes, i.e., bacteria; and eukaryotes, such as fungi, including yeasts, algae, protozoa, molds, and the like, as well as plant cells, both in culture or in planta. Specific bacteria which are susceptible to transformation include members of the Enterobacteriaceae, such as strains of Escherichia coli; Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; Haemophilus influenzae, and yeasts such as Saccharomyces, among others. As used herein, the term microbial host cell encompasses all of these prokaryotic and eukaryotic organisms, including plant cells, both in culture and in planta.
Universal probes can be obtained which hybridize with certain of the fragments of a DNA library, allowing identification and selection (or xe2x80x9cprobing outxe2x80x9d) of the genes of interest, i.e., those nucleotide sequences which encode the polypeptides described as part of the present invention. The isolation of these genes can be performed using techniques which are well known in the art of molecular biology. The isolated genes can be inserted into appropriate vectors for use in the transformation of microbial host cells. In addition, these genes can be subjected to standard nucleic acid sequencing procedures to provide specific information about the nucleotide sequence of the genes encoding the subject polypeptides.
It is now well known in the art that when synthesizing a gene for improved expression in a host cell it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell. For purposes of the subject invention, xe2x80x9cfrequency of preferred codon usagexe2x80x9d refers to the preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. To determine the frequency of usage of a particular codon in a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes expressed by the host cell. It is preferable that this analysis be limited to genes that are highly expressed by the host cell.
Thus, in one embodiment of the subject invention, bacteria, plants, or other cells can be genetically engineered, e.g., transformed with genes from protozoa of the Leishmania spp., to attain desired expression levels of the subject polypeptides or proteins. To provide genes having enhanced expression, the DNA sequence of the gene can be modified to comprise codons preferred by highly expressed genes to attain an A+T content in nucleotide base composition which is substantially that found in the transformed host cell. It is also preferable to form an initiation sequence optimal for said host cell, and to eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and termination of RNA and to avoid sequences that constitute secondary structure hairpins and RNA splice sites. For example, in synthetic genes, the codons used to specify a given amino acid can be selected with regard to the distribution frequency of codon usage employed in highly expressed genes in the host cell to specify that amino acid. As is appreciated by those skilled in the art, the distribution frequency of codon usage utilized in the synthetic gene is a determinant of the level of expression.
Assembly of the genes of this invention can be performed using standard technology known in the art. A structural gene designed for enhanced expression in a host cell can be enzymatically assembled within a DNA vector from chemically synthesized oligonucleotide duplex segments. The gene can then be introduced into the host cell and expressed by means known in the art. Preferably, the protein produced upon expression of the synthetic gene is functionally equivalent to a native protein. According to the subject invention, xe2x80x9cfunctionally equivalentxe2x80x9d refers to identity or near identity of function. A synthetic gene product which has at least one property relating to its activity or function that is similar or identical to a natural protein is considered functionally equivalent thereto.
It is also well known in the art that the nucleotide sequences of the subject invention can be truncated such that certain of the resulting fragments of the original full-length sequence can retain the desired characteristics of the full-length sequence. A wide variety of restriction enzymes are well known by those skilled in the art to be suitable for generating fragments from larger nucleic acid molecules. For example, it is well known that Ba131 exonuclease can be conveniently used for time-controlled limited digestion of DNA. See, for example, Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, pages 135-139. See also Wei et al. (1983) J Biol. Chem. 258:13006-13512. Thus, Ba131 exonuclease (commonly referred to as xe2x80x9cerase-a-basexe2x80x9d procedures) allows for the removal of nucleotides from either or both ends of the subject nucleic acids, consequently generating a wide spectrum of fragments, many of which encode products that are functionally equivalent to the natural polypeptide sequences of the present invention. Labeling procedures are also well known, and the ordinarily skilled artisan could routinely screen the labeled fragments for their hybridization characteristics to determine their utility as probes. For example, it is routine to label nucleic acids for use as specific and selective probes in genetic identification or diagnostic procedures. A person of ordinary skill in the art would recognize that variations or fragments of those sequences, which specifically and selectively hybridize to the DNA of Leishmania spp., could also function as a probe. It is within the ordinary skill of persons in the art, and does not require undue experimentation, to determine whether a segment of the subject nucleic acids is a fragment or variant which specifically and selectively hybridizes in accordance with the subject invention. Therefore, fragments or variants of these nucleic acids can be useful as probes to identify, diagnose, or distinguish Leishmania species.
It would also be recognized that the polynucleotides or peptides of the subject invention can be useful as molecular weight markers in respective nucleic acid or amino acid molecular weight determinations or assays.
In order to obtain a first-generation immunotherapeutic agent according to the subject invention, organisms of the genus Leishmania can be cultivated in synthetic culture medium comprising the ingredients listed in Table 1. In a preferred embodiment, the culture medium is supplemented with 5% fetal bovine serum. Cultivation of the protozoa according to the subject invention is typically carried out at about 30-34xc2x0 C. In a particularly preferred embodiment, cultivation of the protozoa is carried out in the amnastigote stage of its life cycle.
The culture medium comprising the protozoan cells can then be treated in order to inactivate, and preferably kill, the cells. Upon isolation of those cells, the antigenic proteins can be purified therefrom and included in a pharmaceutically acceptable carrier, e.g., buffer solution, to create a second-generation immunotherapeutic agent. Preferably, the cells are inactivated or killed with a non-lysing agent, e.g., TLCK. The antigenic proteins of the present invention are particulate proteins that can be isolated from the cells using accepted methods. In a more specific embodiment the method of creating the second-generation immunotherapeutic agent of the present invention comprises the steps of (1) cultivating to protozoa, preferably in the amastigote stage, in an appropriate culture medium; (2) treating said protozoan cells to inactivate or kill the cells; (3) isolating the treated cells; (4) extracting antigenic proteins from the isolated cells; and (5) formulating the second-generation immunotherapeutic agent composition by combining one or more isolated antigenic proteins with a pharmaceutically acceptable carrier, e.g., phosphate buffered saline (PBS). A preferred pharmaceutically acceptable carrier is a PBS solution having alumina present within the solution.
To cure psoriasis in patients with clinical and histopathological diagnosis of the disease, the first-generation polyvalent immunotherapeutic agent was administered intramuscularly, in the deltoid region, once a month, once every 15 days or once a week according to disease severity, for 7.6xc2x16.0 months on average, at 500 xcexcg/dose.
Furthermore to cure psoriasis a monovalent immunotherapeutic agent with each one of the Leishmania spp. present in the first-generation polyvalent immunotherapeutic agent was used as a subject composition with similar results to the polyvalent immunotherapeutic agent.
Furthermore to cure psoriasis a second-generation immunotherapeutic agent containing the protein fractions isolated by chromatographic means from the crude first-generation immunotherapeutic agent together with 0.1 ml alumina/mg protein was administered intramuscularly in the deltoid region once every 15 days for 3-4 doses at 200 xcexcg/dose in 0.5 ml.